Twitch and tetanic force, contraction and relaxation rate, and resistance to eccentric contraction-induced force loss were significantly decreased in affected dogs. Intriguingly, the time-to-peak tension and half-relaxation time were significantly shortened in affected dogs. Contractile kinetics predicted an unforeseen slow-to-fast myofiber-type switch, which we confirmed at the protein and transcript level. Our study establishes a foundation for studying long-term and late-stage therapeutic interventions in dystrophic canines. The unexpected myofiber-type switch highlights the complexity of muscle remodeling in dystrophic large mammals.Aged dystrophin-null canines are excellent models for studying experimental therapies for Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. To establish the baseline, we studied the extensor carpi ulnaris (ECU) muscle in 15 terminal age (3-year-old) male affected dogs and 15 age/sex-matched normal dogs. Affected dogs showed histological and anatomical hallmarks of dystrophy, including muscle inflammation and fibrosis, myofiber size variation and centralized myonuclei, as well as a significant reduction of muscle weight, muscle-to-body weight ratio and muscle cross-sectional area. To rigorously characterize the contractile properties of the ECU muscle, we developed a novel This article has an associated First Person interview with the first author of the paper. Summary: A slow-to-fast fiber-type switch in dystrophic canine ECU muscle is revealed by contraction kinetics and myosin protein and transcript expression. This highlights the complexity of muscle remodeling in Duchenne muscular dystrophy. Duchenne muscular dystrophy (DMD) is the most severe form of muscular dystrophy, affecting approximately one in every 5000 male births . DMD is There has been tremendous progress in translating experimental therapeutics from animal models to human patients in recent years . HoweverLoss of muscle strength is a primary clinical presentation of DMD patients. Numerous protocols are available for studying muscle physiology in mice. Unfortunately, dystrophin-deficient mdx mice cannot fully recapitulate the contractile features of human patients. For example, the absolute twitch and tetanic muscle forces are maintained at the wild-type level in mdx mice . Methodsin situ protocol for studying force generation in a single intact dog muscle in 2012  and age/sex-matched normal dogs (n=15) using classic anatomical and histopathological methods and our newly developed force assay protocol. The ECU muscle of affected dogs showed significant atrophy and dystrophic pathology. In terms of force measurement, absolute and specific twitch and tetanic forces were significantly decreased, and eccentric contraction-induced force drop was significantly worsened in affected dogs. Kinetic analysis revealed that the time-to-peak tension, half-relaxation time, rate of contraction and rate of relaxation were all significantly reduced in affected dogs. Quantification of the myofiber type revealed an unexpected slow-to-fast fiber-type conversion in the affected ECU muscle. Our findings have laid the groundwork for better utilizing dystrophic canines as a preclinical model for studying DMD pathogenesis and therapy.Given the need to establish the baseline for affected animals that are at the late stage of the disease, we evaluated the ECU muscle in a large cohort of terminal age (3-year-old) affected male dogs  to allow the seamless integration of muscle dissection, mounting, stimulation, force data acquisition, muscle length control and force analysis . The duaTable S1).The assay protocol was developed to accurately quantify the muscle force generated by the ECU muscle from dogs with different body sizes. It included warm up, stimulation condition optimization, tetanic force measurement, twitch force measurement and, finally, ten cycles of eccentric contraction. The warm up was applied to stabilize the muscle for consistent force output during subsequent measurements . OptimalFig. S1).A total of 15 normal and 15 affected male dogs were included in the study . The ECUFig. S2). In the normal muscle, eMyHC was barely detected (<0.2%). In the affected muscle, the number of eMyHC+ myofibers remained low (∼1.6%) but were significantly higher than that of the normal muscle  average rate change and real-time rate change . SegmentFig. S3).The ratio of the twitch force to the maximum tetanic force is often used to characterize the physiological property of a muscle , 2008. ITo comprehensively evaluate force generation, we studied the force-frequency relationship over a broad range of stimulation frequencies  . In all To characterize contraction-induced muscle damage, we evaluated the percentage of the force decrease through ten cycles of eccentric contraction D. The acFig. S4, Table S2; Fig. S4) . Antibody 6H1 has been shown to recognize IIx in mouse, rat and human muscle . In summary, the ECU muscle primarily consisted of type I and IIa fibers . Quantification revealed that the normal ECU was dominated by type I (∼67%), whereas the affected ECU was dominated by type IIa (∼61%), suggesting a slow-to-fast fiber-type switch in dystrophic ECU muscle  . BesidesFig. S4) . Type IIn muscle . HoweverU muscle B. The fiU muscle C. We alsin situ force assay platform and an optimized force assay protocol for studying the contractility of the canine ECU muscle . Importantly, we examined histopathology, muscle force and contractile kinetics of the ECU muscle of a large cohort of terminal-age male dystrophic dogs and age/sex-matched normal dogs . We found characteristic muscle pathologies, such as muscle atrophy, myofiber size variation, degeneration/regeneration, inflammation and fibrosis, in the affected ECU muscle . A physiology assay showed a significant decrease of the absolute and specific twitch and tetanic forces, a significant shortening of the TPT and half-relaxation time, a significant reduction of the contraction and relaxation rate and a significant aggravation of eccentric contraction-induced muscle damage in the affected ECU muscle . Further analysis revealed an unexpected slow-to-fast myofiber-type switch in the affected ECU muscle .In this study, we describe a novel U muscle ; Fig. S2in situ in alive dogs (Table S4) (Table S1). This allows the muscle to reach the best performance. For example, in our previous study in 1.6-year-old dogs, the tetanic force of the affected ECU muscle only reached ∼55 N (Table S3) (Table S4). Given aging-associated disease progression in DMD, we expect muscle force to go down at 3 years of age in affected dogs (Table S5).Accurate measurement of muscle force depends on a robust assay system. We previously reported a manual protocol for studying the force of the ECU muscle able S4) . Howeverable S4) . This noable S3) . In the ted dogs . Howeverted dogs . In the n=15) and age/sex-matched normal dogs (n=15). The absence of dystrophin expression in the affected ECU muscle was confirmed by immunostaining .The average lifespan of dystrophin-deficient dogs is ∼3 years . Quantitstaining A,B. Uponstaining . We alsoTable S4) (Table S4) (Table S4). Collectively, the anatomical data confirm the progression of muscular dystrophy in old affected dogs.We previously observed a statistically insignificant reduction of the ECU muscle weight and pCSA in 1.6-year-old affected dogs (able S4) . Additioable S4) . On the Table S3). We first examined muscle force. In twitch contraction, tetanic contraction and force-frequency assays, the absolute force and pCSA-normalized specific force were all significantly reduced in the affected ECU muscle . These results confirm and expand our previous findings in 1.6-year-old affected dogs . Second, it is well established that the dystrophic muscle undergoes a fast-to-slow, rather than a slow-to-fast, transition . The peak force and TPT of the affected ECU muscle were ∼100 N and ∼714 ms, respectively . Similarly, the average twitch contraction rate, twitch relaxation rate  and tetanic relaxation rate  were all significantly reduced in the affected ECU muscle .The force reduction is not surprising in a dystrophic muscle because dystrophin-null muscle cells undergo necrosis and are replaced by fatty fibrotic tissue that does not have contractile machinery. The reduction of the contraction (or relaxation) rate, we suspect, may relate to the disproportional change of the force and contraction (or relaxation) time. The rate of contraction (or relaxation) is directly proportional to the force but inversely proportional to the time. For example, during tetanic contraction, the peak force and TPT of normal ECU muscle were ∼147 N and ∼831 ms, respectively B,E. Thisectively B,E. ThisFigs S4, S5). We found that the dog ECU muscle had no type IIx and IIb fibers . Importantly, we found the affected ECU muscle mainly consisted of the fast type IIa fiber, whereas the normal ECU muscle mainly consisted of the slow type I fiber  was assigned a slide (tissue) number. The investigators who performed the assay and quantification were blinded to the animal information.The custom-made muscle force assay platform was designed by C.H.H. to accommodate canines with different body weights and sizes . The plax-axis, y-axis and z-axis . The sliding was regulated by the locking mechanism on the linear bushing . The sliding was regulated by the wheel knob-controlled threaded steel rod  connected to a heated circulating water bath . One was placed underneath the animal, and the other was placed on top of the animal throughout the experiment. Heart rate, electrocardiograph, oxygen saturation (SpO2), CO2, blood pressure and body temperature were monitored with a veterinary vital sign monitor  throughout the entire experiment. Vital signs, capillary refill time, mucous membrane color, the palpebral reflex and the pedal reflex were recorded every 15 min.All procedures were performed by the C.H.H. with the assistance of H.T.Y. and J.T. Body hair in the surgical areas was shaved, and skin was disinfected with 70% ethanol. Anesthesia was first induced by intravenous injection of propofol (6 mg/kg), then the subject was intubated, and anesthesia was maintained with 2-4% isoflurane throughout the experiment. The subject was placed on the main platform and positioned in a dorsal recumbency position using foam wedges . Respiration was maintained using a mechanical ventilator  throughout the experiment. The tidal volume was set at 10 ml/min/kg bodyweight, and the breathing rate was set at 12-15 per min to achieve the partial pressure of COA catheter  was inserted into the saphenous vein for intravenous saline infusion . The infusion rate was set to 4 ml/kg/h. The skin between the medial and lateral sides of the neck was disinfected with 70% ethanol, and a 4-6-cm segment of the right carotid artery was surgically exposed. The proximal end of the artery was tied with a 2-0 braided silk suture  to block the blood flow. A small incision was made in the artery, and a silicone tube  was inserted and advanced to the thoracic aorta to measure the central blood pressure. The tube was then secured to the carotid artery using a 2-0 braided silk suture and the skin incision site was closed with a 4-0 braided silk suture .All procedures were performed by C.H.H. with the assistance of H.T.Y. and J.T. Below, we describe the surgical procedure for studying the left ECU muscle. The same procedure can also be adapted to study the right ECU muscle. The entire procedure had five steps, including (1) placement of the artery blood flow probe, (2) exposure of the ECU muscle, (3) determination of the ECU muscle length, (4) exposure of the radial nerve, and (5) fixation of the forelimb with bone pins.To place the blood flow probe, the animal was placed in the left lateral recumbency position. The left forelimb arm was then extended and secured with surgical tape. The medial skin above the elbow was disinfected with 70% ethanol, and the brachial artery was surgically exposed. A 3PS transonic flow probe  was placed around the brachial artery to measure blood flow. The space between the artery and the probe was filled with electrode gel , and the incision site was closed with a 4-0 braided silk suture .To expose the ECU muscle, the animal was carefully repositioned on the right lateral side, and the left forelimb was extended. The skin was disinfected with 70% ethanol, and an incision was made on the lateral side of the left forearm to expose the left ECU muscle. The length of the entire ECU preparation (muscle plus tendon) was measured from the proximal tendon insertion at the medial epicondyle of the humerus to the distal tendon insertion at the carpus. We have previously reported that the tendon length is 16% of the length of the entire ECU preparation . The expThe forearm incision was slightly extended proximately to expose the triceps brachii lateral muscle to expose the radial nerve. The radial nerve was then carefully exposed by retracting the triceps brachii lateral muscle and the triceps brachii accessory caudally. The nerve was then carefully dissected between the triceps brachii accessory and the brachialis muscle, and tied with a 1-0 braided silk suture  at the proximal end and cut close to the collateral radial artery.To secure the forelimb to the dual-axis mount, a stainless-steel bone pin was screwed to the olecranon, and another bone pin was screwed on the radius bone H. FinallTo mount the ECU muscle to the force transducer, the animal was placed in the dorsal recumbency position and supported with foam wedges on the muscle force assay platform G. The fox-axis subcomponent in the tri-axis mount  . BF-35 has been shown to stain all MyHC isoforms except type IIx in dog muscle . In another method, we tested antibody 6H1, a mouse IgM monoclonal antibody . Antibody 6H1 has been shown to specifically stain type IIx fibers in mouse, rat and human muscles . The following secondary antibodies were used in immunostaining: Alexa Fluor 350-conjugated goat anti-mouse IgG2b  for the BA-D5 antibody; Alexa Fluor 594-conjugated goat anti-mouse IgG1  for the SC-71 antibody, FITC-conjugated goat anti-mouse IgM  for the BF-F3 antibody; Alexa Fluor 594-conjugated goat anti-mouse IgG1  for the BF-35 antibody; and Alexa Fluor 488-conjugated goat anti-mouse IgM  for the 6H1 antibody.Following euthanization, the ECU muscle was carefully dissected out and snap-frozen in liquid nitrogen-cooled isopentane in optimal cutting temperature (OCT) compound  for morphological analysis. Cryosections of 10-μm thickness were used for staining. General muscle histopathology was revealed by H&E staining. Fibrosis was examined by Masson trichrome (MTC) staining. Dystrophin was examined by immunofluorescence staining with a monoclonal antibody against the dystrophin C-terminal domain ] . Type II Italy)] . The abo Italy)]  . Laminin was visualized using Alexa Fluor 488-conjugated rabbit anti-goat IgG (H+L) .All antibodies have been validated by the manufacturers. Additional validations were performed in-house in the presence and/or absence of primary and/or secondary antibodies.https://fiji.sc, National Institutes of Health, Bethesda, MD, USA). The minimum Feret diameters of the myofibers were quantified from five random microscopic fields (200× magnification) of a digitalized laminin immunostaining image using MyoVision automated image analysis software  of H&E-stained muscle section using Fiji imaging software (The percentage of fiber-type isoforms was determined from five random microscopic fields (200× magnification) of the MyHC triple immunostaining/laminin co-stained muscle section using Fiji imaging software. Specifically, a photomicrograph from each muscle section was taken with the UV-2A (blue), TRITC (red) and FITC (green) fluorescence filters. The three pictures were uploaded to Fiji imaging software and stacked to an RGB composite image. Different fiber types were distinguished based on intracellular color. Specifically, blue for type I fiber, red for type IIa fiber, magenta for type I/IIa hybrid fiber and green for type IIb fiber. Each type was manually counted using the cell counter plug-in module in the software. The total number of fibers was calculated from the sum of each individual fiber-type count. The percentage of each fiber type was determined by dividing the individual fiber type count by the total number of fibers. All photomicrographs were taken with a Lecia DFC700 color camera using a Nikon E800 microscope.Myosin-enriched muscle lysate was extracted from liquid nitrogen snap-frozen muscle tissue by homogenization in a buffer containing 10% SDS, 1% 0.5 M EDTA and 12.5% 0.5 M Tris-HCl (pH 6.8); supplemented with Halt protease and phosphatase inhibitor cocktail . Next, 1-mm-thick 4% stacking/6% separating SDS polyacrylamide mini gels were prepared according to a modified protocol described previously . SpecifiTable S3). The data were reported as the transcript copy number per ng of cDNA used in the reaction.RNA was extracted from OCT-embedded tissues using an RNeasy Fibrous Tissue kit (Qiagen). The cDNA was generated using a SuperScript IV Kit (Thermo Fisher Scientific) and quantified using a Qubit ssDNA Assay Kit (Thermo Fisher Scientific). Myosin heavy chain transcripts were quantified by digital droplet PCR in a QX200 ddPCR system (Bio-Rad) using ddPCR Supermix for Probes (no dUTP) (Bio-Rad) and custom-designed primers and probes  at different stimulation frequencies was analyzed using one-way ANOVA with Tukey's post-hoc test. All statistical analyses were performed using GraphPad Prism software version 9.1.1 . The difference was considered significant when P<0.05. The datasets used and/or analyzed during the current study are presented in Dataset 1.All data are biological replicates. Data are mean±s.d.. Data were checked with the Shapiro–Wilk test to confirm normality. To compare the statistical significance between normal and affected dogs (two-group comparison), an unpaired Student's


Breast cancer (BC) represents a molecularly and clinically heterogeneous disease. Recent progress in immunotherapy has provided a glimmer of hope for several BC subtypes. The relationship between N6-methyladenosine (m6A) modification and long non-coding RNAs (LncRNAs) is still largely unexplored in BC. Here, with the intention to dissect the landscape of m6A-related lncRNAs and explore the immunotherapeutic value of the m6A-related lncRNA signature, we identified m6A-related lncRNAs by co-expression analysis from The Cancer Genome Atlas (TCGA) and stratified BC patients into different subgroups. Furthermore, we generated an m6A-related lncRNA prognostic signature. Four molecular subtypes were identified by consensus clustering. Cluster 3 preferentially had favorable prognosis, upregulated immune checkpoint expression, and high level of immune cell infiltration. Twenty-one m6A-related lncRNAs were applied to construct the m6A-related lncRNA model (m6A-LncRM). Survival analysis and receiver operating characteristic (ROC) curves further confirmed the prognostic value and prediction performance of m6A-LncRM. Finally, high- and low-risk BC subgroups displayed significantly different clinical features and immune cell infiltration status. Overall, our study systematically explored the prognostic value of the m6A-related LncRNAs and identified a high immunogenicity BC subtype. The proposed m6A-related LncRNA model might serve as a robust prognostic signature and attractive immunotherapeutic targets for BC treatment. The Global Cancer Statistics reported that female breast cancer (BC) surpassed lung cancer as the most diagnosed cancer, with an estimated 2.3 million new cases (11.7%) . BC is gN6-Methyladenosine (m6A) is the most abundant internal epigenetic modification occurring in RNA molecules . The disN6-methyladenosine modification on RNA was involved in various stages of the RNA life cycle, including RNA transcription, processing, splicing, degradation, and RNA translation . m6A reg1. The expression of 24 m6A-related genes was extracted from the TCGA-BRCA, including expression data on writers , readers , and erasers (FTO and ALKBH5). The lncRNAs in the TCGA dataset were identified based on the annotation of Genome Reference Consortium Human Build 38 (GRCh38), and 14,086 lncRNAs were evaluated in the TCGA-BRCA transcriptome matrix. In this study, lncRNAs we identified were composed of eight types of transcripts . In this study, BC samples without complete survival data were excluded from all analyses. The clinical features of TCGA-BRCA samples are presented in A total of 1,109 BC patients and 113 normal paired tissues from the TCGA-BRCA (The Cancer Genome Atlas-Breast Cancer) program were enrolled in our study. The public RNA sequencing and clinical information of patients were obtained from TCGAp < 0.001 in the TCGA-BRCA dataset. The network of m6A-related genes and m6A-related lncRNAs was visualized by Cytoscape software 3.5.1. To explore the prognostic value of m6A-related lncRNAs, univariate Cox regression analysis was conducted to select the m6A-related lncRNAs that were associated with patients’ overall survival (OS). The HR value and Cox p value were calculated. The Wilcoxon signed-rank test was used to compare the expression of m6A-related lncRNAs in BRCA tissues versus normal tissues.The Spearman correlation analysis was applied to screen m6A-related lncRNAs with the criteria of correlation coefficient > 0.4 and To systematically assess the roles and functions of m6A-related lncRNAs, cluster analysis was performed to subgroup BC patients into different groups using the ConsensusClusterPlus package of R software . Based oBased on univariate Cox regression, LASSO regression analysis was analyzed to explore the association between m6A-prognosis lncRNAs and BC risk. The TCGA BRCA patients were randomly divided into training cohort and validation cohort. Twenty-one m6A-related lncRNAs were selected to construct the best risk score model with the “glmnet” package. We calculated the riskScore of each sample with the formula: RiskScore = ExpressionLncRNA1 × CoefficientLncRNA1 + ExpressionLncRNA2 × CoefficientmLncRNA2 + … ExpressionLncRNAn × CoefficientLncRNAn. The samples were divided into high-risk and low-risk groups with the median value of the riskScore. Subsequently, Kaplan–Meier curves and receiver operating characteristic (ROC) curves of 1 year using “timeROC” package were adopted to evaluate the predictive accuracy of the risk model. In addition, univariate Cox regression analysis and multivariate Cox regression analysis were performed to assess the independence of the risk model by regarding risk score as a single characteristic factor. To evaluate the applicability of the model, we combined the clinical traits with riskScore to plot the survival curves.To specifically analyze the immune infiltration, the CIBERSORT analytical tool was adopted to identify the difference of 22 types of immune cells in different BRCA clusters or high- and low-risk groups using the Wilcoxon rank-sum test . A compap value < 0.05 were considered significant enrichments. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) gene sets were performed with GSEA 4.1.0 tool.In our analysis, GSEA was used to determine the biological pathway exchanges in different BRCA clusters. The transcriptome expression data of TCGA-BRCA were analyzed. Gene sets with nominal p value was calculated. Univariate and multivariate Cox regression analyses were conducted to evaluate the independent prognostic value of the risk model based on m6A-related lncRNAs. p-value < 0.05 was considered statistically significant.All analyses were performed using R software (version 4.0.2). The Wilcoxon signed-rank test was adopted for two-group comparison, and the Kruskal–Wallis test was applied for comparison of more than two groups. With the best cutoff value for subgroup stratification, Kaplan–Meier curves were plotted to compare the OS among various subgroups and the log-rank p < 0.001, 491 lncRNAs were significantly correlated with m6A-related regulators and selected for further analysis. To directly visualize the correlation between lncRNAs and m6A-related regulators, a gene network was plotted by Cytoscape. The co-expression network of m6A-related regulators and lncRNAs showed that 14 m6A-related regulators  were significantly related to some lncRNAs, of which RBM15 was the hub regulator   . Recentl < 0.05) . Then, t < 0.05) . MoreoveTo thoroughly explore immune infiltration status in different BC clusters, CIBERSORT algorithms were used to assess the composition of the immune microenvironment. BC patients in cluster 3 tended to have a higher immune score and estimate score and a lower stroma score . We furtTo annotate the potential biological process and pathways between cluster 3 and other clusters, GSEA analysis was computed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signatures based on individual sample expression profiles. Our results showed that various immune response processes were significantly enriched in cluster 3 samples . GO signvia the LASSO analysis (N = 536) and testing cohort (N = 533). The risk score for each group was calculated using the coefficient of 21 lncRNAs , We further investigated whether clinical features were associated with the m6A-LncRM risk score. Overall, the heatmap displayed that BC clusters, immuneScore, patients’ age, and node status between high- and low-risk groups were significantly different . More spr = −0.18, p < 0.001), naive B cells , and plasma cells   . The inf= 0.008) .In the current study, we aimed to identify the prognostic value of m6A-related lncRNAs. The expression of 24 m6A-related genes and 14,086 lncRNAs was extracted from the TCGA-BRCA cohort. Spearman correlation analysis and univariate Cox regression selected 51 m6A-related lncRNAs for further analysis. Consensus clustering analysis revealed a BC subtype (Cluster 3) with best prognosis, and a high expression of immune checkpoints was observed in Cluster 3. CIBERSORT and ESTIMATE analyses further revealed that a high level of immune cell infiltration was found in Cluster 3. GSEA analysis showed that cluster 3 samples were positively enriched in various immune response processes. Furthermore, 21 m6A-related lncRNAs were selected to establish m6A-LncRM for predicting the OS of BC patients. Survival analysis and ROC curves confirmed a promising prognostic value and prediction performance of m6ALncRM. Multivariate Cox regression analysis showed that m6A-LncRM was able to serve as an independent risk factor. Based on the median risk score, the high-risk group had worse survival outcome than the low-risk group across all clinically stratified subgroups. Finally, low- and high-risk BC subgroups displayed significantly different clinical features and immune cell infiltration status. In the present study, we identified a high immunogenicity BC subtype and developed a novel signature m6A-LncRM based on m6A-related lncRNA profiles of BC, which might serve as a predictive biomarker. Furthermore, we also developed a scoring system termed as m6A-LncRM risk score to evaluate individual m6A-related lncRNA patterns, which might be useful in identifying BC patients with high immunogenicity. Our study aimed to investigate the role of m6A in BC from a systems biology point of view, but not focusing on the mechanism of m6A modification in lncRNAs.Existing classification methods of BC subtypes are restricted to mRNA expression analysis . The PAMSince infiltration of various immune cells in the tumor microenvironment was observed in BC, BC was no longer considered as an immunological quiescent tumor type . The advTo improve the accuracy and efficacy on predicting outcome, we constructed a 21-m6A-related lncRNA model and found that this 21-lncRNA signature would independently predict OS in BC patients. Previous studies have attempted to identify an lncRNA-related panel for predicting the risk of tumor recurrence, neoadjuvant treatment response, and survival outcome . Among tin vitro and in vivo experiments are required to confirm the relationship of m6A-related regulators and lncRNA expressions. To explore the suitability and applicability of our model, more independent BC cohorts are needed to verify the prognostic value of the m6A-related lncRNAs model. Since the analyzed data were derived from databases, another limitation is that it is retrospective in nature. Additionally, the mechanism of m6A-related lncRNAs needs to be further investigated to improve the immunotherapy efficacy of BC.Although m6A modifications of LncRNAs are still largely unexplored in BC, our analysis may provide the overall profile of m6A-related lncRNAs in BC. To our knowledge, this is the first report of the m6A-related lncRNA signature in BC. Undeniably, several drawbacks of our study should be noticed that needs to be further explored. Further This study systematically explored the prognostic value of the m6A-related lncRNAs in BC. A high immunogenicity BC subtype was identified that could be a potential candidate for antitumor immunotherapy. The 21-lncRNA model is a robust biomarker that can independently predict OS of TCGA-BRCA datasets and immunotherapy efficacy.The original contributions presented in the study are included in the article/JZ performed most of the analyses and wrote the manuscript. XH and JC designed the study. BS, LL, JD, QS, and QZ performed some of the analyses and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intensive and semi-intensive sheep farming are the two farming systems usually employed in Greece. Several studies have been conducted on the effect of the farming system on sheep milk fatty acid composition. This study simultaneously examines milk basic composition, physicochemical characteristics, fatty acid composition, and nutritional value according to the production system. Bulk tank milk from farms using the intensive and semi-intensive production systems was analyzed. The results show that grazing can improve the fatty acid composition of milk. Thus, pasture-based feeding strategies could improve the fatty acid composition and the nutritional properties of sheep dairy products.Dairy sheep farming is a vital sector of the agricultural economy in Greece. Information on the effect of the farming system on sheep milk characteristics is critical for producing dairy products with improved nutritional value according to the current guidelines for healthy food consumption. This study aimed to investigate the basic composition, physicochemical characteristics, fatty acid composition, and nutritional indices of milk produced in intensive and semi-intensive sheep farms. Bulk tank milk samples from 20 intensive and 20 semi-intensive sheep farms were examined. Sheep in the intensive farms were kept indoors and were fed with roughage, silage, and concentrates. Sheep in the semi-intensive farms were kept indoors during the night and were moved to the pastures during the day. Sheep were fed with roughage, silage, and concentrates in combination with grazing. Milk composition and somatic cell count were determined with automatic analyzers. The physicochemical characteristics were determined with standard laboratory methods. The fatty acid composition was analyzed by gas chromatography. The farming system did not affect milk chemical composition and physicochemical characteristics. However, milk fatty acid composition and nutritional value were significantly improved in milk from farms using the semi-intensive production system, and this favorable effect was attributed to the inclusion of pasture in sheep diet. Sheep breeding has a significant economic impact in the rural mountainous areas of Greece, and thus, there are 6.5 million dairy ewes  that proWith regard to the farming system, Hatziminaoglou et al.  classifiThe effect of the farming system on milk chemical composition, physicochemical characteristics, and fatty acid composition of commercially produced milk has not been extensively studied. Additionally, to our knowledge, no published data have simultaneously studied these groups of variables, enabling the production of milk with improved processing performance and a healthier lipid profile according to the current nutritional recommendations.The objective of this study was to offer updated information on the chemical composition, physicochemical characteristics, fatty acid composition, and nutritional value of sheep milk produced in commercial farms using the intensive and semi-intensive farming system.The study was carried out in dairy sheep farms located in the Regional Unit of Florina, Greece  in the period from late May to late June 2019 and lasted 28 days. The Agricultural Veterinary Bureau of Aminteo supervised the farms. Farms were classified according to the European Food Safety Authority system . Thus, aBulk tank milk samples from 20 intensive and 20 semi-intensive sheep farms were collected. Dairy sheep farms were selected on a convenience basis, i.e., willingness of the farmers to collaborate in the study and to allow sample collection. Milk in the tanks was manually mixed with a plunger to achieve homogeneity before sampling. Samples (2 from each farm) were collected in 50 mL plastic screw-capped flasks, placed in isothermal containers with ice packs, and transported to the laboratory within 4 h following collection. Both samples were collected in the same day. Samples aimed for analyzing Somatic Cell Count (SCC), chemical composition, and physicochemical characteristics were kept at 4 °C and analyzed within 24 h following collection. Sodium azide (0.01 g/100 mL) was added as a preservative to the milk samples destined to determine Somatic Cell Count. Samples aimed for the determination of fatty acid composition were kept at −20 °C until analyzed.Milk composition  was determined by infrared analysis (FTIR interferometer), using a Milkoscan FT6000 Analyzer . Milk moisture content was calculated by deducting the total solids content from 100, and milk ash content was calculated by deducting fat, protein, and lactose contents from the total solids content. The ratio fat content/protein content was also determined. Somatic Cell Count (SCC) was determined with a Fossomatic 5000 instrument , and the obtained data were log-transformed to normalize the distribution.Before analysis, the samples were placed in a heated water bath to reach room temperature and, following that, they were thoroughly mixed by gentle inversion of the sample container multiple times without causing frothing. Milk pH was measured using a glass electrode with a built-in temperature sensor  in a Crison GLP 21 pH-meter , which was calibrated with standard buffer solutions at pH 4.0 and 7.0 according to the manufacturer’s instructions. The electrical conductivity of the samples was measured by a GLP 31 conductometer  using a Sodium Ion-Selective Electrode 9650, calibrated with 147 μS/cm, 1413 μS/cm, and 12.88 mS/cm buffer solutions. Refractive index and Brix value were determined using a digital refractometer  set at 20 °C.v/v) that contained 0.01% (w/v) of t-butyl-hydroxytoluene (BHT) to prevent fatty acid oxidation during extraction, according to the Bligh and Dyer method [Fatty acid composition was determined by gas chromatography (GC) as described by Papaloukas et al. . Brieflyr method . Fatty ar method  of the IThe milk fatty acid profile was used to calculate the following indices related to healthy fat consumption. In addition, all nutritional indices were used to assess the nutritional value of milk and other dairy products in various studies.The hypocholesterolemic/hypercholesterolemic fatty acid ratio (h/H) ratio was calculated according to the formula reported by Chen and Liu :h/H = (CThe atherogenicity (AI) and thrombogenicity (TI) indices were calculated according to the following formulae proposed by Ulbricht and Southgate :AI = [C1The health-promoting index (HPI), was proposed by Chen et al. , was calThe Desirable Fatty Acid (DFA) index was calculated according to the formula of Rhee  as follop-values were <0.05.One-factor variance analysis was employed for the comparisons between the farming systems. Correlations between SCC and milk composition were calculated as Pearson’s correlations. SPSS software  was used for data analysis. The results were considered to be significant when the p ≥ 0.05) ash, fat, and protein content and a lower lactose content compared to intensively produced milk (p ≥ 0.05) in the fat/protein ratio between milk samples from the two production systems. The ratio was similar (1.199) to the one calculated considering the fat and protein content of milk from dairy ewes in Greece [Milk from the semi-intensive system had a higher but non significantly different  in relation to the two production systems. Bulk tank somatic cell count is the first and most important index used by farmers and the dairy industry to assess udder health in the flocks and milk hygiene because increased somatic cell counts affect milk production and cheese yield. Although the European Union (Regulation 853/2004) [5 vs. 4.50 × 105 cells/mL, respectively).Milk somatic cell count also did not differ (53/2004)  has set 53/2004) , sheep m53/2004)  proposed53/2004)  also repp ≥ 0.05) in relation to the two production systems, as expected  reported by Pappa et al. . HoweverMilk pH is used as an indirect index for the microbiological quality of milk, and thus, titratable acidity is usually determined. The other three variables, i.e., electrical conductivity, refractive index, and Brix value, are not frequently determined. Electrical conductivity is used as a rapid method for detecting subclinical mastitis in individual animals . Refractcis-9) and linoleic (C18:2 n-6 cis) acids were the predominant fatty acids in the monounsaturated and polyunsaturated lipid classes, respectively. The levels of linolenic (C18:2 n-6) and α-linolenic (C18:3 n-3) acids did not differ statistically (p ≥ 0.05) in milk form the intensive and the semi-intensive system. Grass contains elevated levels of α-linolenic (C18:3 n-3) acid, and this can explain the elevated levels of α-linolenic acid and its biohydrogenation intermediates such as conjugated linoleic acid (CLA) (C18:2 cis-9 trans-11), vaccenic acid (C18:1 trans 11), and stearic acid (C18:0) in the milk from ewes raised using the semi-intensive system [p < 0.001) content of CLA, which possesses anti-carcinogenic, anti-atherogenic, anti-obesity, and anti-diabetic properties [p < 0.001) levels of arachidonic acid (C20:4 n-6), whereas there were no differences in the levels of eicosatrienoic (C20:3 n-3), eicosapentenoic , and docosapentenoic (C22:5 n-3) acids in the milk from both production systems. Sheep milk does not contain high levels of long-chain PUFA due to the extensive biohydrogenation of these acids in the rumen [n-3), and conjugated linoleic acid (CLA) (C18:2 cis-9 trans-11). Furthermore, green plants are the primary dietary sources of n-3 PUFA in ruminants, and increased dietary levels of n-3 PUFA can improve fatty acid composition in ruminant milk and meat [Sheep milk fatty acid composition is presented in y p ≥ 0.0 in milk operties . Milk anoperties ,41. Withhe rumen  in generhe rumen . Forage he rumen , pastureand meat . Furtherand meat , the ratThe pattern of the fatty acid profile for milk from the intensive production system was similar to the one reported by De Renobales et al.  for dairp < 0.001) in the milk from the intensive system, whereas the contents of MUFA and PUFA were significantly higher (p < 0.001) in the milk from ewes on the semi-intensive system. However, there were no differences (p ≥ 0.05) in the medium-chain triglycerides (MCT) content in relation to the two production systems. Sheep milk is rich in MCT, which are important from a therapeutic point of view since they are easily absorbed and can be used in cases of nutrient malabsorption or undernourishment [p < 0.001) in the milk from farms using the semi-intensive system. Chen and Liu [p < 0.001) in milk from the semi-intensive production system than in milk from the intensive production system. In a recent review by Chen and Liu [p < 0.001) in the milk from the semi-intensive system. The reported range for TI was 1.00–2.72 [p < 0.001) in the milk from ewes in the semi-intensive farms. Bonano et al. [p < 0.001) in the milk from the semi-intensive production system. Atti et al. [The effect of the production system on the lipid quality of milk is presented in rishment . The h/H and Liu  reported and Liu . AI was  and Liu , it was .00–2.72 . Both AI.00–2.72  and lowe.00–2.72 . Lower v.00–2.72  reportedo et al.  reportedo et al. . Finallyi et al.  also repi et al. ,53.The data obtained in this study showed no effect of the farming system on the basic composition and physicochemical characteristics of commercially produced milk in farms using the intensive or the semi-intensive production system. However, milk from the semi-intensive production system had significantly improved fatty acid composition and lipid quality nutritional indices in relation to milk produced in the intensive farms. Differences in the composition of fatty acids and in the lipid quality indices were attributed to the inclusion of pasture in sheep diet. This nutritional information could add value to the produced dairy products and create product differentiation.


Infectious bronchitis virus (IBV) leads to huge economic losses in the poultry industry worldwide. The high levels of mutations of IBV render vaccines partially protective. Therefore, it is urgent to explore an effective antiviral drug or agent. The present study aimed to investigate the in vivo anti-IBV activity of a mixture of plant essential oils (PEO) of cinnamaldehyde (CA) and glycerol monolaurate (GML), designated as Jin-Jing-Zi.The antiviral effects were evaluated by clinical signs, viral loads, immune organ indices, antibody levels, and cytokine levels. The infection rates in the PEO-M (middle dose) and PEO-H (high dose) groups were significantly lower than those in the prevention, positive drug, and PEO-L (low dose) groups. The cure rates in the PEO-M and PEO-H groups were significantly higher than those in the prevention, positive drug, and PEO-L groups, and the PEO-M group had the highest cure rate of 92.31%. The symptom scores and IBV mRNA expression levels were significantly reduced in the PEO-M group. PEO significantly improved the immune organ indices and IBV-specific antibody titers of infected chickens. The anti-inflammatory factor levels of IL-4 and IFN-γ in the PEO-M group maintained high concentrations for a long time. The IL-6 levels in the PEO-M group were lower than those in prevention, positive drug, and PEO-L groups.The PEO had remarkable inhibition against IBV and the PEO acts by inhibiting virus multiplication and promoting immune function, suggesting that the PEO has great potential as a novel anti-IBV agent for inhibiting IBV infection.The online version contains supplementary material available at 10.1186/s12917-022-03183-x. Infectious bronchitis virus (IBV) is a pathogenic avian coronavirus. Infectious bronchitis (IB) caused by IBV has brought serious economic losses to the poultry industry that result from the decline of weight gain, decreased egg production, eggshell quality, feed efficiency, false layers, and secondary bacterial infections , 2. At p50) /50 μL [Plant essential oils (PEO) are natural volatile aromatic substances extracted from various parts of plants as part of plant secondary metabolism . PEO had) /50 μL . A previ) /50 μL . Grifths) /50 μL .Houttuynia cordata had an inhibitory effect on IBV in vitro and in vivo [Sambucus nigra [, Mentha piperita, Thymus vulgaris, Desmodium canadense extracts [Astragalus polysaccharides [Glycyrrhiza radix [Forsythia suspensa [The use of plant extracts as an alternative or supplement to treatment or prevention strategy for IBV has not been extensively investigated. Only a few investigations have been performed to verify the anti-IBV activity of plant extracts –23. A mi in vivo . Additious nigra , Mentha extracts , Astragacharides , Glycyrrza radix , and Forsuspensa  have shosuspensa –22, withIn the present study, we investigated the in vivo antiviral effect of a mixture PEO of cinnamaldehyde (CA) and glycerol monolaurate (GML) against avian coronaviruses IBV. To the best of our knowledge, it is the first study to examine the effects of these plant preparations on IBV replication. We chose to study these two essential oil ingredients owing to their known antiviral properties. Cinnamon oil and CA significantly inhibited the growth of influenza virus and respiratory syncytial virus (RSV) . PreviouHerein, the clinical efficacy evaluation, immune organ index, humoral immunity, and cellular immunity as well as the amount of viral RNA were used to assess the prevention and treatment effects of PEO. We sought to identify the antiviral effect of PEO containing CA and GML against IBV in vivo. Our results showed that the PEO has a good effect on inhibiting IBV infection, suggesting that the PEO has great potential as a novel anti-IBV agent.p < 0.01), with the challenge control group scoring significantly (p < 0.01) higher than the prevention and drug groups. At 5 days of administration, scores in the positive drug group and all PEO groups were significantly (p < 0.01) lower than that in the challenge control group. The PEO-M and PEO-H groups had the lowest symptom scores, which were significantly (p < 0.01) lower than the prevention, positive drug, and PEO-L groups. Two and three days after drug withdrawal, symptom scores were reduced to 0 in the PEO-M group, and to 2 in the prevention, positive drug, PEO-L, and PEO-H groups, all of which were significantly (p < 0.01) lower than those in the challenge control group.The clinical symptoms of IBV-infected chicks were observed and scored at various time intervals Fig. . The symThe chicks successively appeared depression, decreased appetite, increased water intake, frequent head shaking, hanging wings, coughing, mouth breathing, and tracheal rales at 3 days post-challenge (dpc). At 5 dpc, the morbidity reached a high level, accounting for more than half of the incidence. The positive drug and PEO groups were given drug treatment, respectively. The cure rate of each group was observed on the 7th day after drug administration Table . The resp < 0.01) than those of positive drug and prevention groups . Furthermore, after 5 days of drug withdrawal, the PEO-M group had lower viral loads in the trachea and kidney than the PEO-L and PEO-H groups, and the mRNA expression levels of IBV in PEO groups were significantly (p < 0.05) lower than those in the positive drug group. When compared to the challenge control, PEO supplementation resulted in a decrease in viral mRNA levels at the majority of time points.To assure the inhibitory effect of PEO, the viral mRNA levels in the trachea and kidney of different groups were measured by real-time quantitative PCR (qPCR) at 2 and 5 days of drug withdrawal. No virus was detected in the trachea and kidney of birds of the blank control group. The mRNA expression levels of IBV in the trachea and kidney of the challenge control group were increased remarkably (p < 0.05) different compared to the blank control group. The spleen index of prevention, positive drug and PEO-M groups increased significantly (p < 0.01) compared with the challenge control group. The thymus and bursa of Fabricius indexes of the challenge control group were significantly (p < 0.01) lower than the blank group. Compared with the challenge group, the thymus and bursa of Fabricius increased in the prevention and drug groups, and the difference was very significant (p < 0.01). Furthermore, the prevention and PEO-M groups had higher organ indices than other drug groups. After 5 days of drug withdrawal, the spleen, thymus, and bursa of Fabricius indexes in the blank control group were significantly (p < 0.01) higher than those in the challenge control group. Spleen, thymus, and bursa indices were elevated in the prevention and drug groups, with highly significant differences compared with the challenge control group (p < 0.01). The organ index of the PEO-M group was higher than those of the positive drug group, PEO-L, and PEO-H groups.The spleen, thymus, and bursa of Fabricius were collected from 5 birds per group on the 2nd and 5th day after drug withdrawal and were weighed individually to calculate relative organ weights Fig. . At 2 dap < 0.01) higher than those of the blank and preventive groups. Chickens in the challenge control group developed higher antibody titers than those in the blank control group 3 days after administration. On day 5 of administration, the antibody levels of the challenge control group were significantly (p < 0.01) higher than that of the blank control group. Moreover, the antibody titers in the different dose PEO groups, positive drug group, and preventive group were significantly (p < 0.01) higher than those in the challenge control group. The antibody levels in the PEO-M and positive drug groups were higher than those in other drug groups. After 2 and 5 days of drug withdrawal, the antibody titers in the prevention, positive drug, and PEO groups were significantly (p < 0.01) higher than those in the challenge control group, and the titers in the PEO-M and PEO-H groups were also significantly (p < 0.05) higher than that in the PEO-L group. During the experiment, compared with the blank control group, the challenge control group had significantly (p < 0.01) higher antibody titers. These results suggested that PEO could improve the humoral immune response in a dose-dependent manner.To examine whether PEO has adjuvant effects on the humoral immune response in birds, antibody titers were determined by ELISA. As shown in Fig. p < 0.01 or p < 0.05) higher than those in the challenge control group. The same tendency was observed at 2 and 5 days of drug withdrawal. After administration, compared with the challenge control group, IL-6 expression levels in the prevention, positive drug, and PEO groups were notably diminished (p < 0.01). The levels of IL-6 in PEO groups were considerably downregulated alike to prevention and positive drug groups, and it was significantly decreased in the PEO-M and PEO-H groups at day 5 of drug withdrawal.To assess the effects of PEO on cytokines in IBV infected chickens, the levels of type II interferon IFN-γ, anti-inflammatory cytokine IL-4, and proinflammatory cytokine IL-6 were investigated during the experiments Fig. . The resIBV is a Gamma coronavirus with multiple identified strains that continue to mutate and recombine, resulting in complex and diverse genotypes and serotypes of IBV. It is challenging to prevent and control IBV infection due to poor cross-protection between the vaccine and field strains. Thus, developing an effective antiviral therapy is a critical approach in combating IBV infection. We investigated the inhibitory effects of the synergistic blend of PEO including CA and GML on IBV in this study. Our findings indicated that the PEO used in this study has an antiviral effect on IBV within a range of doses. Especially PEO could significantly inhibit virus proliferation and improve immune function at 1.0 mL/L. To the best of our knowledge, this is the first application of PEO (including CA and GML) for the inhibition of IBV in China, establishing the groundwork for the development of anti-IBV infection drugs.Sambucus nigra extract inhibits IBV in the early stages of infection and suggests that it does so by damaging the structure of the virus, possibly making it non-infectious [Alium sativum, Houttuynia cordata, and Sambucus nigra may be associated with direct inactivation of viral envelope structures that are required for adsorption to or entry into host cells or may lyse the IBV envelope [Houttuynia cordata-treated viruses were less infectious to chickens, suggesting the direct virucidal effect of Houttuynia cordata [p < 0.01) lower than that in the challenge control group  lower than that in the challenge control group  higher than those in the preventive and positive drug groups. The titers in the PEO-M and PEO-H groups were significantly (p < 0.05) higher than those in the PEO-L group. These results showed that PEO could improve the humoral response in chickens, which was consistent with previous findings [In the fight against infection, antibody level is an important indicator of humoral immunity. The humoral response is thought to be a crucial component of the immune response that protects against IBV. It has been reported that high antibody levels are associated with protection against IBV infection . In addifindings , 47.distinctly defined the antiviral action of IFN-γ against viruses of various genetic nature including Newcastle disease virus (NDV) and Marek’s disease virus (MDV) [An excessive inflammatory response, as a vital immune response of the organism, can cause a dramatic increase in cytokines in the organism, causing a disruption of the immune system and ultimately irreversible damage to the host. It has been reported that coronaviruses have developed strategies to evade the host innate mechanism by increasing the expression of proinflammatory cytokines and decreasing the production of type1 interferons , 49. Furus (MDV) , 53. Chius (MDV) , 55. HowIn the present study, the potential antiviral activity of the PEO was investigated using the widespread and pathogenic Mass41 strain of the virus. However, there are multiple genotypes and serotypes of IBV strains, and co-infection of multiple serotypes is common in the field. Hence, it is necessary to observe the inhibitory effect of PEO on other serotypes of IBV strains or PEO on the simultaneous challenge of multiple serotypes of IBV strains in the future, in order to explore a potentially effective alternative approach that can combat all serotypes of IBV strains.In conclusion, the PEO mixture of CA and GML had remarkable inhibition against IBV and the PEO acts by inhibiting virus multiplication and promoting immune function, suggesting that the PEO has great potential as a novel anti-IBV agent for the inhibition of IBV infection. Our study also provides a reference for effective treatments and prevention of other animal coronaviruses. Further studies are needed to better understand the likely mechanism of this inhibition, underlying immune mechanisms causing these effects, and to assess the ability of PEO to provide a safe and effective alternative method against avian IBV in commercial production facilities.50) of IBV M41 strain was examined and calculated according to the Reed-Muench method [honeysuckle, scutellaria, and forsythia as main components, the positive drug control, was obtained from Beijing Ruixianong Technology Development Co., Ltd., Beijing, China. 1 mL of the positive drug is equivalent to 1.5 g of the original drug, and 1 mL of plant essential oil used in this experiment contains 0.2 g of active ingredients.The pathogenic IBV strain M41 (Lot number AV1511) was purchased from the China Institute of Veterinary Drug Control and propagated in specific pathogen free (SPF) 10-day-old embryonating chicken eggs . The 50% tracheal organ culture infection dose . The PEO-L, PEO-M, and PEO-H groups were given orally 0.5 mL/L, 1.0 mL/L, and 2.0 mL/L PEO respectively. The positive drug group received only Shuanghuanglian Oral Liquid at 0.7 mL/L does following the manufacturer’s instructions. All chicks were given water and feed ad libitum from 8 to 12 days old. Birds in the PEO-L, PEO-M, PEO-H, and positive drug groups were given drugs in drinking water for 5 consecutive days from 13 to 17 days old. All birds were placed into molded plastic separate isolators and fed ad libitum. The birds in challenge groups and the black control group were fed in two separate and nonadjacent rooms to avoid cross-infection.140 3-day-old Yellow feather broiler chicks, purchased from Guangxi Fufeng Agricultural and Animal Husbandry Group Co., Ltd. , were randomly assigned to seven groups of 20 birds each, namely blank control, challenge control, prevention, positive drug, PEO-L, PEO-M, and PEO-H groups . The viral RNA levels were assessed by qPCR targeting the N gene as our previous description . AbsolutAt 2 and 5 days of drug withdrawal, five chickens per group were individually weighed to determine body weight. The spleen, thymus, and bursa of Fabricius of five birds from each group were then collected and weighed separately to calculate relative organ weights, which were represented as immune organ indices. Organ index = organ weight (milligram)/body weight (gram) .Serum samples were harvested from the birds before administration (5 dpc), on days 3 (8 dpc) and 5 (10 dpc) of administration, the 2nd (12 dpc), and 5th day (15 dpc) after drug withdrawal for the antibody titer assay. Total serum IBV-specific antibody was tested using an indirect enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.Serum samples were collected before administration, on the 3rd and 5th day after administration, days 2 and 5 of drug withdrawal. The levels of anti-inflammatory cytokine IFN-γ and IL-4, as well as proinflammatory cytokine IL-6 concentrations, were evaluated by ELISA Kits  following the instructions.n = 5) has been regarded as an experimental unit. GraphPad Prism 8 and Microsoft Excel were used for the statistical analysis of all data, including the determination of the calculation of the means and the geometric mean with 95% CI. The clinical signs score curve was obtained using GraphPad Prism 8. Statistical analyses of viral loads, organ indices, IBV-specific antibody titers, and cytokine concentrations were performed by a one-way analysis of variance (ANOVA) using SPSS 24.0 software. Post-hoc analyses were carried out to identify the significant difference between treatment means using Tukey’s test. P values less than 0.05 (p < 0.05) were considered significant and those less than 0.01 were considered highly significant.The replicate Additional file 2:Supplementary Table 2 .The basic diet composition and nutrient level.)Additional file 3:Supplementary Fig. 1. Pathological changes of trachea in chicken. (A)Blank control. (B) Challenge control. (C)Prevention. (D) Positive drug. (E) PEO-L. (F) PEO-M. (G)PEO-H.)


The long-term health consequences of coronavirus disease 2019 (COVID-19) remain largely unclear. This study aimed to apply the Mendelian randomization (MR) design to estimate the causal associations between COVID-19 and ten cardiovascular conditions.Single-nucleotide polymorphisms (SNPs) associated with COVID-19 were used as instrumental variables to estimate the causal effect of COVID-19 on ten cardiovascular conditions. The random-effects inverse-variance weighted (IVW) method was conducted for the main analyses with a complementary analysis of the weighted median and MR-Egger approaches.P = 0.045) and heart failure  with similar estimates in weighted median regressions. No directional pleiotropic effects were observed in both funnel plots and MR-Egger intercepts.In the IVW analysis, genetically predicted COVID-19 was suggestively associated with major coronary heart disease events (OR 1.081; 95% CI 1.007–1.16; Our findings provide direct evidence that patients infected with COVID-19 are causally associated with increased risk of cardiovascular disease, especially for major coronary heart disease events and heart failure. The outbreak of the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is rapidly evolving as a worldwide health crisis. As of October 3, 2021, this worldwide health crisis has directly resulted in more than 212 million confirmed cases with a mortality of 2.3%, which means more than 4.8 million people directly died of COVID-19. Up to now, a lot of studies have revealed a significant observational association between cardiovascular diseases and COVID-19 (Mendelian randomization (MR) is a recently emerged technique and conceptually similar to prospective randomized controlled trials, which can be used to assess the causality between the risk factor and particular disease –5 due toIn our study, all the summary data was obtained from publicly published studies. Their institutional review committee has approved their design and data in respective studies. Therefore, no further sanction was required in the present study. Two-sample MR –9 was usP < 5 × 10−8) (r2 < 0.005), and only SNPs in both the exposure and outcome GWAS datasets were included in our analysis.Summary-level genetic data for COVID-19 were acquired from results of the Genetics of Mortality in Critical Care (GenOMICC) genome-wide association study (GWAS) , which i × 10−8) . To idenhttps://www.finngen.fi/en/access_results). To be able to determine the differential cardiovascular risk associated with COVID-19, we analyzed a broad range of cardiovascular diseases, including aortic aneurysm, thrombo-embolic diseases (deep vein thrombosis and pulmonary embolism), and other cardiovascular diseases . Due to no individual patient data being available, we used summary data for cardiovascular diseases.Corresponding data for cardiovascular diseases were obtained from the FinnGen project , which was used to extract the summary data set for GWAS of cardiovascular diseases. FinnGen study launched in Finland in the autumn of 2017 is a unique study that combines genome information with digital health care data. The FinnGen study is an unprecedented global research project representing one of the largest studies of this type. This data freeze consists of 176,899 individuals, almost 17,000,000 variants and 2,444 disease endpoints  were excluded, identified by MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) method (P < 5 × 10−3 (a = 0.05/10 outcomes) was used in our analysis. Associations with P-values between 5 × 10−3 and 0.05 were considered suggestive evidence of associations, which required further confirmation.In the principal analyses, an inverse-variance weighted meta-analysis with a random-effects model was used . As a fi) method . In a se) method  and MR-E) method  were useI2 statistics and corresponding p-value derived from Cochran's Q test were used to quantify heterogeneity between estimates from different diseases. Random-effect model meta-analyses were used in our study to pool instrumental variable estimates in the effect of COVID-19 on different diseases. R-based “meta” package was used in all of our meta-analyses.To probe the total effect of COVID-19 on cardiovascular disease and cerebrovascular disease, a meta-analysis was made. All exposure-specific MR analyses were performed for each cardiovascular disease from the FinnGen project and then were meta-analyzed to generate the pooled estimates for COVID-19 on the risk of cardiovascular disease and cerebrovascular disease, separately. The Based on a reasonable request, the related data and statistical coding can be obtained from the corresponding author. The MR software packages TwosampleMR (0.5.6) and R version 4.0.3 (2020-10-10)  were used in our study , 16.P < 5 × 10−8). None of the eight SNPs had previously been reported to play a part in any pathway.The mean age of the 2,244 patients with COVID-19 included in the present analysis was 57.3 years and 69.74% were men. As shown in P = 0.029; P = 0.045; There was suggestive evidence of a positive association between genetically predicted COVID-19 and major coronary heart disease events  is characterized by a long incubation period, high transmission, and diverse clinical manifestations , which hn = 13,464) and cardiovascular conditions , there is a causal effect of COVID-19 on the risk of major coronary heart disease event and heart failure, conferring 8.1 and 4.9% increased risk, respectively.MR is a recently emerged technique, which is conceptually similar to prospective randomized controlled trials (RCT) because all the inherited genetic variants are determined prior to the disease onset. Our present MR analysis can provide a good solution to avoid potential confounding factors and assess the causal effect of COVID-19 on cardiovascular disease. Based on summary statistics from the newest GWAS studies for COVID-19  can be found in the article/Ethical approval was not provided for this study on human participants because in our study, all the summary data was obtained from publicly published studies. Their institutional review committee has approved their design and data in respective studies. Therefore, no further sanction was required in the present study. The patients/participants provided their written informed consent to participate in this study.Y-LC designed the study and revised the manuscript. MJ finished the analysis. H-JC helped with the use of software. L-MJ, H-JC, and MJ finished the draft of this manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Glioblastoma is a malignant intracranial tumor with indispensable growth. Identification of biomarkers associated with the progression of tumors could benefit the clinical therapy of and improve patient’s survival. miR-411 has been reported to play a role in other cancers, while its function in glioblastoma has been explored in the present study. The expression of miR-411 was evaluated in glioblastoma tissues (collected from 108 glioblastoma patients) and cells by polymerase chain reaction. The clinical significance of miR-411 was estimated with a series of statistical analyses. The biological function of miR-411 in the cellular processes of glioblastoma was assessed by cell counting kit 8 and Transwell assay. The expression of miR-411 was significantly reduced in glioblastoma, which was associated with the Karnofsky Performance Score (KPS) and Isocitrate dehydrogenase 1 (IDH1) status of patients. miR-411 was identified as an independent prognostic indicator that correlated with the poor prognosis of patients. miR-411 suppressed the growth, migration, and invasion of glioblastoma cells via modulating signal transducer and activator of transcription 3 (STAT3). miR-411 participated in the development of glioblastoma and function as a prognostic biomarker. miR-411 functions as a tumor suppressor, which provides a novel potential therapeutic target for glioblastoma. Glioblastoma is a kind of aggressive and lethal brain tumor in humans ,2. SurgiThe functions of microRNAs (miRNAs) have been widely reported in various human cancers, especially miRNAs with abnormal expression. miRNAs are short non-coding RNAs with a length of 18–25 nt. Previously, miRNAs have been evidenced to regulate the growth, differentiation, migration, and invasion of various cells. These cellular processes are also crucial factors for the development of tumors. In glioblastoma, several miRNAs were identified as potential biomarkers for disease onset and progression, such as miR-589-3p, miR-758-5p, and miR-185 ,6,7. miRThe abnormal expression of miR-411 in glioblastoma implies its potential of acting as a biomarker in glioblastoma. The expression level of miRNAs was different in dissimilar cancers. The downregulation of miR-411 has been observed in renal cell carcinoma and breast cancer ,13, and The patient enrollment was conducted from 2012 to 2014, and this study had obtained approval from the Ethics Committee of the First Affiliated Hospital of Xiamen University . One hundred and eight patients diagnosed with glioblastoma and underwent surgery were included in this study. The participants had never received any chemotherapy or radiotherapy before surgical treatment and signed informed consent. All patients were followed up for 5 years to summarize their survival information. Paired glioblastoma tissues and normal tissues were collected during surgery and frozen in liquid nitrogen for the following analyses.2 [U343, T98, A172, and LN229 cells were used as the typical glioblastoma cell lines, and the NHA cells were selected as the normal consultation cell lines. All cells were purchased from ATCC and maintained in the DMEM culture medium with 10% FBS (Sigma Aldrich). Cell culture was performed in a cell culture flask at 37°C with 5% CO2 .Cells were transfected with miR-411 mimic, miR-411 inhibitor, or negative controls when they arrived the logarithmic growth phase with the employment of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions . Transfe−ΔΔct method with U6 as the reference gene [Collected tissues and treated cells were lysed to extract total RNA with TRIzol reagent. cDNA was generated from RNA with the help of reverse transcriptase . Then, cDNA was used for the quantification of miR-411 with the SYBR Green Master kit (Qiagen). The relative expression of miR-411 (primer sequences: forward 5ʹ-CCATGUAUGUAACACGGUCCAC-3ʹ and reverse: 5ʹ-GGUUAGUGGACCGGTCACC-3ʹ) was calculated with the 2nce gene ,19.The wild-type and mutant-type STAT3 vectors were established by cloning binding sites or mutant sites into the pmirGLO plasmid. The co-transfection of established vectors and miR-411 mimic, miR-411 inhibitor, or negative controls was conducted with the Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The luciferase activity of STAT3 was detected with the Dual-luciferase Reporter Assay System with the normalization to Renilla [Treated cells were seeded into 96-well plates and supplied with a complete culture medium. The seeded plates were incubated in an incubator at 37°C for a certain period of time. CCK8 reagent was added to each well, and the incubation was continued for 2 h. The absorbance at 450 nm was detected by a microplate reader to evaluate cell growth .For the assessment of cell migration and invasion, treated cells were seeded into the upper chamber of 24-well transwell plates (BD Bioscience). The upper chamber was precoated with Matrigel (Corning) before the invasion assay. The seeded cells were supplied with a serum-free medium, and the bottom chamber was filled with a complete medium with FBS as the chemoattractant. The migration and invasion assay was conducted at 37°C for 24 h, then the migrated and invaded cells were stained with crystal violet and analyzed with an optical microscope (Olympus) .P < 0.05.All data were obtained from independent triplicate experiments and expressed as a mean value ± SD. Data were analyzed using the SPSS 20.0 software. The difference between the two groups was estimated by Student’s t-test, and one-way ANOVA was used to evaluate the differences among multiple groups. The clinical significance of miR-411 was assessed with the Chi-square test, Kaplan–Meier analysis, and Cox regression analysis. The results were considered to be statistically significant when in vitro cell experiments.miR-411 was speculated to play a role in the progression of glioblastoma. The expression of miR-411 was evaluated in glioblastoma tissues and cells. The specific role of miR-411 in glioblastoma was estimated with a series of statistical analyses and P < 0.001, P < 0.001, In glioblastoma tissues, miR-411 was dramatically reduced in comparison with normal tissues (P = 0.018) and IDH1 (P = 0.019) status was demonstrated with the help of the Chi-square test  and a wild type of IDH1. The significant association of miR-411 expression with the KPS  together with KPS  and IDH1 status  of patients (Additionally, the results of Kaplan–Meier analysis showed that patients in the low miR-411 expression group possessed a shorter lifetime than patients in the high miR-411 expression group (Log-rank patients .Table 2.P < 0.001, P < 0.001, P < 0.05, P < 0.01, A172 and LN229 cells were transfected with miR-411 mimic, which significantly enhanced the expression level of miR-411 (P < 0.001, P < 0.01, P < 0.001, Moreover, the regulatory effect of miR-411 was also observed in the migration and invasion of glioblastoma cells. In the presence of miR-411 overexpression, the number of migrated cells plummeted, which surged in the presence of miR-411 silencing (P < 0.001, P > 0.05, In the STAT3 wild-type vector that contains the binding sites between miR-411 and STAT3 , the lucIdentification of novel molecules participating in the progression of glioblastoma and possessing the ability to regulate cell processes is of great interest in cancer research. In previous studies, the function of miRNAs has been widely investigated. For example, miR-1238 was reported to play a vital role in the treatment of glioblastoma as the fact that miR-1238 mediated the chemoresistance of glioblastoma and CAV1/EGFR was revealed to be the direct target during its functioning . miR-449The dysregulation of miR-411 was previously found in the expression profile of differentially expressed miRNAs in glioblastoma compared with non-tumor brain tissues by Skalsky et al. . KPS is miR-411 was also indicated to be involved in the development of many other cancers and regulate various major cellular processes. For instance, the downregulation of miR-411 was found in renal cell carcinoma, which showed a significantly inhibitory effect on cell growth and migration and induced effect on cell apoptosis . OtherwiMoreover, the mechanism underlying the function of miR-411 could benefit the clinical application of miR-411 prognostic biomarker and tumor suppressor function. Numerous molecules have been confirmed as targets of miR-411 during its biological effect on various tumor progressions. Previously, the interaction between miR-411 and STAT3 has been demonstrated to be the mechanism underlying the tumor suppressor role of miR-411 in cervical cancer . STAT3 wThe downregulation of miR-411 in glioblastoma was correlated with the disease development and clinical outcome of patients. miR-411 serves as a tumor suppressor, which might be a novel therapeutic target of glioblastoma.